Molecular mechanisms of signal transducer and activator of transcription (STAT) protein function in hematopoiesis

Hematopoietic growth factors play important roles in regulating blood cell growth and development in vivo. In this work, we investigated the signaling mechanisms of two growth factors with clinical significance, erythropoietin (Epo) and granulocyte colony-stimulating factor (G-CSF). Epo is essential for the survival, proliferation and differentiation of red blood cell progenitors, while G-CSF plays an important role in controlling mature neutrophil production. To identify which amino acid(s) and/or motif in EpoR is responsible for cell survival, wild type or mutant EpoR isoforms were transfected into the growth factor-dependent 32D cell line. Proliferation and apoptosis assays demonstrated that an EpoR isoform that lacks intracellular tyrosine residues and is truncated after 321 amino acids in the cytoplasmic tail (EpoR 1-321) mediates Epo-dependent cell survival. Furthermore, in absence of fetal calf serum (FCS), Epo signaling through wild type or mutant receptors supported anti-apoptosis, but not proliferation during 72 hours in response to Epo. To investigate the signaling pathway by which EpoR regulates cell survival, a dominant negative Stat5b (dnStat5b) isoform was generated and coexpressed with EpoR in stable cell lines. Expression of dnStat5b causes a significant induction of apoptosis in the presence of Epo in cells expressing EpoR 1-321, indicating that Stat5 is essential for survival signaling through tyrosine independent sequences in the EpoR. In a second project to investigate G-CSF signaling, we studied mechanisms by which G-CSF regulates the expression of PU.1, an important transcription factor in myeloid and B cell development. We demonstrated, by immunoblot and real time RT-PCR, that PU.1 is induced by G-CSF ex vivo as well as in vivo. To test whether G-CSF signaling through Stat3 is required for PU.1 regulation, the upstream region of the PU.1 gene was analyzed for potential Stat3 binding motifs. Four potential sites were identified; chromatin immunoprecipitations demonstrated that G-CSF activated Stat3 binds to 3 of the 4 binding motifs. In addition, PU.1 induction by G-CSF was completely abrogated in bone marrow from hematopoietic conditional Stat3 knockout mice. These results indicate an important role for Stat3 in G-CSF-dependent PU.1 gene regulation. Collectively, our works demonstrate that Stat protein play important and diverse roles in hematopoietic growth factor signaling. ^

Cellular mechanisms of operant and classical conditioning

The ability to associate a predictive stimulus with a subsequent salient event (i.e., classical conditioning) and the ability to associate an expressed behavior with the consequences (i.e., operant conditioning) allow for a predictive understanding of a changing environment. Although they are operationally distinct, there has been considerable debate whether at some fundamental level classical and operant conditioning are mechanistically distinct or similar. Feeding behavior of Aplysia (i.e., biting) was chosen as the model system and was successfully conditioned with appetitive forms of both operant and classical conditioning. The neuronal circuitry responsible for feeding is well understood and is suitable for cellular analyses, thus providing for a mechanistic comparison between these two forms of associative learning. ^ Neuron B51 is part of the feeding circuitry of Aplysia and is critical for the expression of ingestive behaviors. B51 also is a locus of plasticity following both operant and classical conditioning. Both in vivo and in vitro operant conditioning increased the input resistance and the excitability of B51. No pairing-specific changes in the input resistance were observed following both in vivo and in vitro classical conditioning. However, classical conditioning decreased the excitability of B51. Thus, both operant and classical conditioning modified the threshold level for activation of neuron B51, but in opposite directions, revealing key differences in the cellular mechanisms underlying these two forms of associative learning. ^ Next, the cellular mechanisms underlying operant conditioning were investigated in more detail using a single-cell analogue. The single-cell analogue successfully recapitulated the previous in vivo and in vitro operant conditioning results by increasing the input resistance and the excitability of B51. Both PKA and PKC were necessary for operant conditioning. Dopamine appears to be the transmitter mediating the reinforcement signal in this form of conditioning. A D1 dopamine receptor antibody revealed that the D1receptor localizes to the axon hillock, which is also the region that gives the strongest response when iontophoresing dopamine. ^ The studies presented herein, thus, provide for a greater understanding of the mechanisms underlying both of these forms of associative learning and demonstrate that they likely operate through distinct cellular mechanisms. ^

Molecular mechanisms of prostacyclin receptor signaling: The intracellular domains in G protein coupling

To better understand the mechanisms of how the human prostacyclin receptor (1P) mediates vasodilation and platelet anti-aggregation through Gs protein coupling, a strategy integrating multiple approaches including high resolution NMR experiments, synthetic peptide, fluorescence spectroscopy, molecular modeling, and recombinant protein was developed and used to characterize the structure/function relationship of important segments and residues of the IP receptor and the α-subunit of the Gs protein (Gαs). The first (iLP1) and third (iLP3) intracellular loops of the IP receptor, as well as the Gαs C-terminal domain, relevant to the Gs-mediated IP receptor signaling, were first identified by observation of the effects of the mini gene-expressed corresponding protein segments in HEK293 cells which co-expressed the receptor and Gαs. Evidence of the IP iLP1 domain interacted with the Gαs C-terminal domain was observed by fluorescence and NMR spectroscopic studies using a constrained synthetic peptide, which mimicked the IP iLP1 domain, and the synthetic peptide, which mimicked Gαs C-terminal domain. The solution structural models and the peptide-peptide interaction of the two synthetic protein segments were determined by high resolution NMR spectroscopy. The important residues in the corresponding domains of the IP receptor and the Gαs predicted by NMR chemical shift mapping were used to guide the identification of their protein-protein interaction in cells. A profile of the residues Arg42 - Ala48 of the IP iLP1 domain and the three residues Glu392 ∼ Leu394 of the Gαs C-terminal domain involved in the IP/Gs protein coupling were confirmed by recombinant proteins. The data revealed an intriguing speculation on the mechanisms of how the signal of the ligand-activated IP receptor is transmitted to the Gs protein in regulating vascular functions and homeostasis, and also provided substantial insights into other prostanoid receptor signaling. ^

Investigation of the molecular mechanisms of endometrial hyperplasia

While there is considerable information on the molecular aberrations associated with the development of endometrial cancer, very little is known of changes in gene expression associated with its antecedent premalignant condition, endometrial hyperplasia. In order to address this, we have compared the level of expression of components of the IGF-I signaling pathway in human endometrial hyperplasia to their level of expression in both the normal pre-menopausal endometrium and endometrial carcinoma. We have also characterized the molecular characteristics of endometrial hyperplasia as it occurs in a murine model of hormone-dependent tumorigenesis of the female reproductive tract. ^ There was a significant and selective increase in the expression of the IGF-I Receptor (IGF-IR) in both human hyperplasia and carcinoma as compared to the normal endometrium. The receptor was also activated, as judged by increased tyrosine phosphorylation. In addition, in hyperplasia and carcinoma there is activation of the downstream component Akt. The expression of the PTEN tumor suppressor is decreased in a subset of subjects with hyperplasia and in all of the carcinomas. The simultaneous loss of PTEN expression and increased IGF-IR activation in the hyperplastic endometrium was associated with an increased incidence of endometrial carcinoma elsewhere within the uterus. In the rodent hyperplasia, there was a significant increase in the expression and activation of Akt that appears to be attributable to a marked increase in the expression of IGF-II. ^ Our studies have demonstrated the pathologic proliferation of both the human and rodent endometrium is linked to a marked activation of the Akt pathway. However the cause of this dysregulation is different in the human disease and the animal model. In rodents, hyperplasia is linked to increased expression of one of the ligands of the IGF-IR, IGF-II. In humans the IGF-I receptor itself is upregulated and activated. Additional activation of the Akt pathway via the suppression of PTEN activity, results in conditions that are associated with the marked increase in the probability of developing endometrial cancer. Our data suggests that increased activity of the IGF-I pathway plays the key role in the hyperproliferative state characteristic of endometrial hyperplasia and cancer.^

Mechanisms of heterosynaptic modulation of transmission in Aplysia

Heterosynaptic plasticity has received considerable attention as a means to induce and maintain cell-wide, as opposed to synapse-specific, learning-related modifications. Modulatory neurotransmitters are thought to provide the attentional and motivational state for memory formation. However, the cellular and molecular mechanisms mediating the effects of most of these modulators on synaptic plasticity and learning remain unclear. A well established system for the study of heterosynaptic plasticity is the Aplysia sensorimotor synapse, which is subject regulation by at least two neuromodulators, serotonin (5-HT) and FMRFa. ^ 5-HT engages multiple second messenger cascades to induce short- and long-term facilitation (STF and LTF, respectively) of synaptic transmission. One mechanism proposed to be involved in STF is mobilization of synaptic vesicles from a storage pool to a releasable pool. To investigate this hypothesis, we examined the involvement of the protein synapsin, a central element in the regulation of the storage pool of vesicles in nerve terminals, in STF. 5-HT induced phosphorylation of synapsin and modified its subcellular distribution via PKA and p42/44 MAPK. Electrophysiological experiments and computer simulations suggested that synapsin can support heterosynaptic plasticity by regulating vesicle mobilization. ^ FMRFa induce short- and long-term synaptic depression in Aplysia . Long-term depression (LTD) correlates with morphological changes, the mechanisms of which remain elusive. LTD is also transcription- and translation-dependent, but little is known about the genes expressed and their regulation. We investigated the role of protein degradation via the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by the transcription factor CREB2, which is generally regarded as a transcription repressor. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions. ^ These and additional studies on the interaction of the 5-HT and FMRFa-activated pathways suggest that different neuromodulators, by activating several and sometimes overlapping signaling cascades, can exercise bidirectional control on synaptic gain and information processing.^

The molecular mechanisms of sensing nucleoside analogue-induced DNA damage

Nucleoside analogues are antimetabolites effective in the treatment of a wide variety of solid tumors and hematological malignancies. Upon being metabolized to their active triphosphate form, these agents are incorporated into DNA during replication or excision repair synthesis. Because DNA polymerases have a greatly decreased affinity for primers terminated by most nucleoside analogues, their incorporation causes stalling of replication forks. The molecular mechanisms that recognize blocked replication may contribute to drug resistance but have not yet been elucidated. Here, several molecules involved in sensing nucleoside analogue-induced stalled replication forks have been identified and examined for their contribution to drug resistance. ^ The phosphorylation of the DNA damage sensor, H2AX, was characterized in response to nucleoside analogues and found to be dependent on both time and drug concentration. This response was most evident in the S-phase fraction and was associated with an inhibition of DNA synthesis, S-phase accumulation, and activation of the S-phase checkpoint pathway (Chk1-Cdc25A-Cdk2). Exposure of the Chk1 inhibitor, 7-hydroxystaurosporine (UCN-01), to cultures previously treated with nucleoside analogues caused increased apoptosis, clonogenic death, and a further log-order increase in H2AX phosphorylation, suggesting enhanced DNA damage. Ataxia-telangiectasia mutated (ATM) has been identified as a key DNA damage signaling kinase for initiating cell cycle arrest, DNA repair, and apoptosis while the Mre11-Rad50-Nbs1 (MRN) complex is known for its functions in double-strand break repair. Activated ATM and the MRN complex formed distinct nuclear foci that colocalized with phosphorylated H2AX after inhibition of DNA synthesis by the nucleoside analogues, gemcitabine, ara-C, and troxacitabine. Since double-strand breaks were undetectable, this response was likely due to stalling of replication forks. A similar DNA damage response was observed in human lymphocytes after exposure to ionizing radiation and in acute myelogenous leukemia blasts during therapy with the ara-C prodrug, CP-4055. Deficiencies in ATM, Mre11, and Rad50 led to a two- to five-fold increase in gemcitabine sensitivity, suggesting that these molecules contribute to drug resistance. Based on these results, a model is proposed for the sensing of nucleoside analogue-induced stalled replication forks that includes H2AX, ATM, and the Mre11-Rad50-Nbs1 complex. ^

Reactive oxygen species-dependent mechanisms of N-(4-hydroxyphenyl)retinamide (4HPR)-induced apoptosis in human carcinoma cells

4HPR is a synthetic retinoid that has shown chemopreventive and therapeutic efficacy against premalignant and malignant lesions including oral leukoplakia, ovarian and breast cancer, and neuroblastoma. 4HPR induces apoptosis in various cancer cells and production of reactive oxygen species (ROS) has been suggested as a possible cause underlying these effects. However, the mechanisms governing these effects by 4HPR are not fully elucidated. In this study, we explored the mechanisms of 4HPR-induced ROS increase and apoptosis in human cancer cells. ^ First, we identified genes modulated by 4HPR using oligonucleotide gene expression arrays and found that they fall into specific functional canonical pathways and gene networks using Ingenuity Pathways Analysis®. Further analysis has shown that 4HPR induced up-regulation of Endoplasmic Reticulum (ER)-related genes such as Heat shock proteins 70 and 90 and the transcriptional factor, GADD153. These findings were validated using quantitative real-time PCR. ^ Second, we found that 4HPR induced extensive ER stress evidenced by dilation of the ER and endoribonuclease-mediated splicing and activation of the transcriptional factor, XBP-1. In addition, 4HPR induced the up-regulation of various ER stress-related genes and their protein products, as well as cleavage and activation of the ER specific Caspase-4. Concomitantly with XBP-1 splicing, all of these effects were dependent on ROS generation by 4HPR. Furthermore, chemical inhibition and RNA interference studies revealed a novel pro-apoptotic role for HSP70/A1A in 4HPR-mediated apoptosis. ^ Third, we observed rapid activation of the small GTPase Rac by 4HPR which was upstream of ROS generation. Inhibition of Rac activity or silencing of its expression by RNA interference inhibited ROS generation and apoptosis induction by 4HPR. siRNA targeting PAK1 and expression of a dominant negative Rac, decreased 4HPR-mediated ROS generation, while expression of a constitutive active Rac increased basal and 4HPR-induced ROS generation and PARP cleavage. Furthermore, metastatic cancer cells exhibited higher Rac activation, ROS generation, and cell growth inhibition due to 4HPR exposure compared to their primary cancer cell counterparts. ^ These findings provide novel insights into 4HPR-mediated ROS generation and apoptosis induction and support the use of ROS inducing agents such as 4HPR against metastatic cancer cells. ^

Molecular mechanisms of response in gastrointestinal stromal tumors treated with imatinib mesylate

Gastrointestinal stromal tumors (GIST) represent 80% of sarcoma arising from the GI tract. The inciting event in tumor progression is mutation of the kit or, rarely, platelet derived growth factor receptor-α (PDGFR) gene. These mutations encode ligand independent, constitutively active proteins: Kit or PDGFR. ^ These tumors are notoriously chemo and radio resistant. Historically, patients with advanced disease realized a median overall survival of 9 months. However, with modern management of GIST with imatinib mesylate (Novartis), a small molecule inhibitor of the Kit, PDGFR, and Abl tyrosine kinases, patients now realize a median overall survival greater than 30 months. However, almost half of patients present with surgically resectable GIST and the utility of imatinib in this context has not been prospectively studied. Also, therapeutic benefit of imatinib is variable from patient to patient and alternative targeted therapy is emerging as potential alternatives to imatinib. Thus, elucidating prognostic factors for patients with GIST in the imatinib-era is crucial to providing optimal care to each particular patient. Moreover, the exact mechanism of action of imatinib in GIST is not fully understood. Therefore, physicians find difficulty in accurately predicting which patient will benefit from imatinib, how to assess response to therapy, and the time at which to assess response. ^ I have hypothesized that imatinib is tolerable and clinically beneficial in the context of surgery, VEGF expression and kit non-exon 11 genotypes portend poor survival on imatinib therapy, and imatinib's mechanism of action is in part due to anti-vascular effects and inhibition of the Kit/SCF signaling axis of tumor-associated endothelial cells. ^ Results herein demonstrate that imatinib is safe and increases the duration of disease-free survival when combined with surgery. Radiographic and molecular (namely, apoptosis) changes occur within 3 days of imatinib initiation. I illustrate that non-exon 11 mutant genotypes and VEGF are poor prognostic factors for patients treated with imatinib. These findings may allow for patient stratification to emerging therapies rather than imatinib. I show that imatinib has anti-vascular effects via inducing tumor endothelial cell apoptosis perhaps by abrogation of the Kit/SCF signaling axis. ^

Role of T cell response during vaccine immunity to tuberculosis

Tuberculosis remains one of the leading causes of death in man due to a single infectious agent. An estimated one-third of the world's population is infected with the causative agent, Mycobacterium tuberculosis (Mtb), despite the availability of the widely used vaccine, BCG. BCG has significantly varying protection rates with the lowest level of protection seen with the most common form of TB, adult pulmonary TB. Thus, numerous studies are being conducted to develop a more efficient vaccine. The ideal candidate vaccine would possess the ability to induce a solid and strong Th1 response, as this is the subset of T cells primarily involved in clearance of the infection. A novel vaccine should also induce such a response that may be recalled and expanded upon subsequent infection. Our group has introduced a mutant of a virulent strain of Mtb which lacks a component of the immunogenic antigen 85 complex (Ag85). Our vaccine, ΔfbpA, does not secrete the fibronectin binding protein Ag85A, and this has shown to lead to its attenuation in both murine macrophages and mice. Previous studies have also proven that ΔfbpA is more protective in mice than BCG against virulent aerosol challenge with Mtb. This study addresses the mechanisms of protection observed with ΔfbpA by phenotyping responding T cells. We first evaluated the ability of dendritic cells to present the mycobacteria to naïve T cells, an in vitro mock of primary immunization. We also measured the response of primed T cells to macrophage-presented mycobacteria to interpret the possible response of a vaccinated host to a boost. We concluded that ΔfbpA can elicit a stronger Th1 response compared to BCG in vitro, and further observed that this enhanced response is at least partly due to the presence of proteins encoded by a region of the genome absent in all strains of BCG. Finally, we observed this heightened Th1 response in the mouse model after primary vaccination and a virulent aerosol challenge. The cytolytic T cell response was also measured after virulent challenge and was found to be superior in the ΔfbpA-treated group when compared to the BCG group. ^

Mechanisms of transcription inhibition by 8-amino-adenosine

Nucleoside analogs are a class of chemotherapeutic agents with tremendous utility in treating viral infections and cancers. Traditional nucleoside analogs are DNA-directed. However, there is a new group of nucleoside analogs that induce cell death by a direct effect on RNA synthesis. The adenosine analog, 8-chloroadenosine, is incorporated into RNA and is currently in clinical trials. Another congener, 8-amino-adenosine has demonstrated toxicity in multiple myeloma cell lines. Like other nucleoside analogs, 8-amino-adenosine must be metabolized to its triphosphate to elicit a cytotoxic effect. Furthermore, 8-amino-adenosine causes a decline of the intracellular ATP pool and inhibits mRNA poly(A) adenylation. ^ Because of the previously known adenosine analog mechanism as well as the scope of the RNA directed nucleoside analog field, I hypothesized there are multiple mechanisms of transcription inhibition mediating 8-amino-adenosine-induced cell death. Prior to investigating these mechanisms, cell death by 8-amino-adenosine was characterized. 8-Amino-adenosine activates PARP cleavage and induces the caspase cascade. 8-Amino-adenosine increases Annexin V binding and the mitochondrial membrane permeability in wild-type MEF cells. In BAX/BAK deficient MEF cells, 8-amino-adenosine decreases the mitochondrial membrane permeability and induces autophagy. ^ Once cell death was characterized, the mechanisms of 8-amino-adenosine transcription inhibition were assessed. It was established that 8-aminoadenosine treatment causes 8-amino-ATP accumulation and decreases the intracellular ATP concentration, resulting in RNA synthesis inhibition. Several other mechanisms are identified. First, a relationship between ATP decline by 8-amino-adenosine or other known ATP synthesis inhibitors and RNA synthesis is established indicating that effects on cellular bioenergy, regardless of the mechanism of ATP decline, can decrease RNA synthesis. Second, 8-aminoadenosine treatment decreases the phosphorylation of serine residues on the RNA polymerase II C-terminal domain which regulates transcription initiation and elongation. Third, evidence is provided to demonstrate 8-amino-ATP is a substrate for RNA synthesis. Fourth, 8-amino-ATP is incorporated at the 3'-terminal position leading to chain termination. Finally, in vitro transcription assays show that 8-amino-ATP may compete with ATP to decrease de novo mRNA synthesis. Overall, this work demonstrates 8-amino-adenosine is a cytotoxic nucleoside analog that functions to inhibit RNA transcription through multiple mechanisms. ^

A movement from traditional malarial chemoprophylaxis to the use of intermittent preventive treatment as a method of protection for children diagnosed with sickle-cell disease: A proposal for consideration based on a systematic review of the literature

Approximately 200,000 African children are born with sickle-cell anemia each year. Research has shown that individuals with hemoglobin disorders, particularly sickle-cell anemia, have increased susceptibility to contracting malaria. Currently it is recommended that patients diagnosed with sickle-cell anemia undergo malaria chemoprophylaxis in order to decrease their chances of malarial infection. However, studies have shown that routine administration of these drugs increases the risk of drug resistance and could possibly impair the development of naturally acquired immunity. Clinical trials have shown intermittent preventive treatment (IPT) to be an effective method of protection against malaria. The objective of this report was to review previously conducted clinical trials that study the effects of intermittent preventive treatment on malaria and anemia in infants and children. Based on the review, implications for its appropriateness as a protective measure against malaria for infants and children diagnosed with sickle-cell disease were provided.^ The 18 studies reviewed were randomized controlled trials that focused on IPT’s effect on malaria (7 studies), anemia (1 study), or both (8 studies). In addition to these 16, one study looks at IPT’s effect on molecular resistance to malaria, and another study is a follow-up to a study in order to review IPT’s potential to cause a rebound effect. The 18 th study in this review specifically looks at IPT’s protective efficacy in children with SCA. The studies in this report were restricted to randomized controlled trials that have been performed from 2000 to 2010. Reports on anemia were included to illustrate possible added benefits of the use of IPT specific to burdens associated with SCA other than malaria susceptibility. The outcomes of these studies address several issues of concern involving the administration of IPT: protective efficacy (in reference to age, seasonal versus perennial malaria regions, and overall effectiveness against malaria and anemia), drug resistance, drug rebound effect, drug side-effects, and long-term effects. Overall, these showed that IPT has a significant level of protective efficacy against malaria and/or anemia in children. More specifically, the IPT study evaluating children diagnosed with sickle-cell anemia proved IPT to be a more effective method of protection than traditional chemoprophylaxis. ^

IMMUNOLOGICAL MECHANISMS OF EXTRACORPOREAL PHOTOPHERESIS IN CUTANEOUS T CELL LYMPHOMA AND GRAFT VERSUS HOST DISEASE

IMMUNOLOGICAL MECHANISMS OF EXTRACORPOREAL PHOTOPHERESIS IN CUTANEOUS T CELL LYMPHOMA AND GRAFT VERSUS HOST DISEASE Publication No.___________ Lisa Harn-Ging Shiue, B.S. Supervisory Professor: Madeleine Duvic, M.D. Extracorporeal photopheresis (ECP) is an effective, low-risk immunomodulating therapy for leukemic cutaneous T cell lymphoma (L-CTCL) and graft versus host disease (GVHD), but whether the mechanism(s) of action in these two diseases is (are) identical or different is unclear. To determine the effects of ECP in vivo, we studied regulatory T cells (T-regs), cytotoxic T lymphocytes (CTLs), and dendritic cells (DCs) by immunofluorescence flow cytometry in 18 L-CTCL and 11 GVHD patients before and after ECP at Day 2, 1 month, 3 months, and 6 months. In this study, ECP was effective in 12/18 L-CTCL patients with a 66.7% overall response rate (ORR) and 6/11 GVHD patients with a 54.5% ORR. Prior to ECP, the percentages of CD4+Foxp3+ T cells in 9 L-CTCL patients were either lower (L-CTCL-Low, n=2) or higher (L-CTCL-High, n=7) than normal. Five of the 7 GVHD patients had high percentages of CD4+Foxp3+ T cells (GVHD-High). Six of 7 L-CTCL-High patients had >80% CD4+Foxp3+ T cells which were correlated with tumor cells, and were responders. Both L-CTCL-High and GVHD-High patients had decreased percentages of CD4+Foxp3+ and CD4+Foxp3+CD25- T cells after 3 months of treatment. CD4+Foxp3+CD25+ T cells increased in GVHD-High patients but decreased in L-CTCL-High patients after 3 months of ECP. In addition, numbers of CTLs were abnormal. We confirmed that numbers of CTLs were low in L-CTCL patients, but high in GVHD patients prior to ECP. After ECP, CTLs increased after 1 month in 4/6 L-CTCL patients whereas CTLs decreased after 6 months in 3/3 GVHD patients. Myeloid (mDCs) and plasmacytoid DCs (pDCs) were also low at baseline in L-CTCL and GVHD patients confirming the DC defect. After 6 months of ECP, numbers and percentages of mDCs and pDCs increased in L-CTCL and GVHD. MDCs were favorably increased in 8/12 L-CTCL responders whereas pDCs were favorably increased in GVHD patients. These data suggest that ECP is favorably modulating the DC subsets. In L-CTCL patients, the mDCs may orchestrate Th1 cell responses to overcome immune suppression and facilitate disease regression. However, in GVHD patients, ECP is favorably down-regulating the immune system and may be facilitating immune tolerance to auto-or allo-antigens. In both L-CTCL and GVHD patients, DCs are modulated, but the T cell responses orchestrated by the DCs are different, suggesting that ECP modulates depending on the immune milieu. _______________

Molecular Mechanisms of Vascular Disease in Patients with Rare Variants in MYH11

Thoracic aortic aneurysms and dissections (TAAD) are the primary disease affecting the thoracic ascending aorta, with an incidence rate of 10.4/100,000. Although about 20% of patients carry a mutation in a single gene that causes their disease, the remaining 80% of patients may also have genetic factors that increase their risk for developing TAAD. Many of the genes that predispose to TAAD encode proteins involved in smooth muscle cell (SMC) contraction and the disease-causing mutations are predicted to disrupt contractile function. SMCs are the predominant cell type in the ascending aortic wall. Mutations in MYH11, encoding the smooth muscle specific myosin heavy chain, are a rare cause of inherited TAAD. However, rare but recurrent non-synonymous variants in MYH11 are present in the general population but do not cause inherited TAAD. The goal of this study was to assess the potential role of these rare variants in vascular diseases. Two distinct variants were selected: the most commonly seen rare variant, MYH11 R247C, and a duplication of the chromosomal region spanning the MYH11 locus at 16p13.1. Genetic analyses indicated that both of these variants were significantly enriched in patients with TAAD compared with controls. A knock-in mouse model of the Myh11 R247C rare variant was generated, and these mice survive and reproduce normally. They have no structural abnormalities of the aorta or signs of aortic disease, but do have decreased aortic contractility. Myh11R247C/R247C mice also have increased proliferative response to vascular injury in vivo and increased proliferation of SMCs in vitro. Myh11R247C/R247C SMCs have decreased contractile gene and protein expression and are dedifferentiated. In fibroblasts, myosin force generation is required for maturation of focal adhesions, and enhancers of RhoA activity replace enhancers of Rac1 activity as maturation occurs. Consistent with these previous findings, focal adhesions are smaller in Myh11R247C/R247C SMCs, and there is decreased RhoA activation. A RhoA activator (CN03) rescues the dedifferentiated phenotype of Myh11R247C/R247C SMCs. Myh11R247C/R247C mice were bred with an existing murine model of aneurysm formation, the Acta2-/- mouse. Over time, mice carrying the R247C allele in conjunction with heterozygous or homozygous loss of Acta2 had significantly increased aortic diameter, and a more rapid accumulation of pathologic markers. These results suggest that the Myh11 R247C rare variant acts as a modifier gene increasing the risk for and severity of TAAD in mice. In patients with 16p13.1 duplications, aortic MYH11 expression is increased, but there is no corresponding increase in smooth muscle myosin heavy chain protein. Using SMCs that overexpress Myh11, we identified alterations in SMC phenotype leading to excessive protein turnover. All contractile proteins, not just myosin, are affected, and the proteins are turned over by autophagic degradation. Surprisingly, these cells are also more contractile compared with wild-type SMCs. The results described in this dissertation firmly establish that rare variants in MYH11 significantly affect the phenotype of SMCs. Further, the data suggests that these rare variants do increase the risk of TAAD via pathways involving altered SMC phenotype and contraction. Therefore, this study validates that these rare genetic variants alter vascular SMCs and provides model systems to explore the contribution of rare variants to disease.

Mechanisms of multidrug resistance: Differences between natural and acquired adriamycin -resistant human colon carcinoma cells

Mechanisms of multidrug resistance (MDR) were studied in two independent MDR sublines (AdR1.2 and SRA1.2) derived from the established human colon carcinoma cell line LoVo. AdR1.2 was developed by long-term continuous exposure of the cells to adriamycin (AdR) in stepwise increments of concentration, while SRA1.2 was selected by repetitive pulse treatments with AdR at a single concentration. In this dissertation, the hypothesis that the mechanism of drug resistance in SRA1.2 is different than that in AdR1.2 is tested. While SRA1.2 demonstrated similar biological characteristics when compared to the parental LoVo, AdR1.2 exhibited remarkable alterations in biological properties. The resistance phenotype of AdR1.2 was reversible when the cells were grown in the drug-free medium whereas SRA1.2 maintained its resistance for at least 10 months under similar conditions. Km and Vmax of carrier-mediated facilitated diffusion AdR transport were similar among the three lines. However, both resistant sublines exhibited an energy-dependent drug efflux. AdR1.2 appeared to possess an activated efflux pump, and a decreased nucleus-binding of AdR, whereas SRA1.2 showed merely a lower affinity in binding of AdR to the nuclei. Southern blot analysis showed no amplification of the MDR1 gene in either of the two resistant subclones. However, Western blot analysis using the C219 monoclonal antibody against P170 glycoprotein detected a Mr 150-kDa plasma protein (P150) in AdR1.2 but not in SRA1.2 or in the parental LoVo. In vitro phosphorylation studies revealed that P150 was a phosphoprotein; its phosphorylation was Mg$\sp{2+}$-dependent and could be enhanced by verapamil, an agent capable of increasing intracellular AdR accumulation in AdR1.2 cells. The phosphorylation studies also revealed elevated phosphorylation of a Mr 66-kDa plasma membrane protein that was detectable in the AdR1.2 revertant and in AdR1.2 when verapamil was present, suggesting that hyperphosphorylation of the Mr 66-kDa protein may be related to the reversal of MDR. SDS-PAGE of the plasma membrane protein demonstrated overproduction of a Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa protein was not immunoreactive with C219, but its absence in the AdR1.2 revertant and the parental LoVo suggests that it is an MDR-related plasma membrane protein. In conclusion, the results from this study support the author's hypothesis that the mechanisms responsible for "Acquired" and "Natural" MDR are not identical. ^